A popular approach in molecular biology involves the introduction of specific mutations in cloned genes for the analysis of phenotypes. Shortle, D., J. Biol. Chem. 264, 5315-5318 (1989). This reverse-genetic approach, employing site-directed mutagenesis, has facilitated the elucidation of structure-function relationships for a large number of genes. Such methods have also been successfully used for the design of desired characteristics into gene products for use in research and its applications. In some instances, such experiments have revealed intricacies of functional organization that were not apparent from the primary sequence or expression patterns. Matthews, B., Biochemistry 26, 6885-6887 (1987).
Methods of site-directed mutagenesis have evolved rapidly since the initial description of this concept. Smith, M., Annv. Rev. Genet. 19, 423-462 (1985). A common feature of the available methods is the use of synthetic oligonucleotides carrying the desired changes in the nucleotide sequence at the site of mutagenesis. This "mutant" oligonucleotide is incorporated into the sequence of interest by replacing the normal sequences with the designed oligonucleotide. This is accomplished by in vitro enzymatic DNA synthesis. A second step that requires the propagation and resolution of mutant and wild-type sequences in bacteria can greatly influence the rate of mutagenesis. Recently, the use of specially selected strains of E. coli that will allow enrichment of mutant molecules has improved the efficiency of mutagenesis. Kunkel, T. A., Proc. Natl. Acad. Sci. USA 82, 480-492 (1985).
Both the efficiency and the speed of mutagenesis have been improved by the introduction of methods based on the Polymerase Chain Reaction (PCR). Saiki, R. K. et al., Science 239, 487-491 (1986). Several methods based on PCR have been described that allow the introduction of mutations into one of the two primers used for the amplification of DNA. Higuchi, R. et al., Nucl. Acids Res. 16, 7351-7367 (1988); Valette, F. et al., Nucl. Acid Res. 17, 723-733 (1989); Kadowaki, H. et al., Gene 76, 161-166 (1989); Dubau, L. et al., Nucl. Acids Res. 17, 2873 (1989). However, these methods are limited to the mutagenesis of the sequences located at the termini of the amplified sequences. Other methods that permit the modification of amplified sequences via the use of primers that overlap two non-contiguous sequences during amplification, have been described. However, such methods are limited by the nature of the sequence overlaps and require multiple steps. Ho, S. N. et al., Gene 77, 51-59 (1989); Yon, J. et al., Nucl. Acids Res. 1-7, 4895 (1989); Mole, S. E. et al., Nucl. Acids Res. 17, 3319 (1989); Kammann, M. et al., Nucl. Acids Res. 17, 5404 (1989).
A method that would permit the incorporation of desired mutations at any site in the target of PCR amplification would greatly expand the utility of PCR mutagenesis.